Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells.

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 microm and 0.5-1.5 microm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.     

Dual Functions for Connexins: Cx43 Regulates Growth Independently of Gap Junction Formation

Connexins, the family of proteins that form vertebrate gap junctions, have key roles during development and in the adult. Previously, the physiological actions of connexins have been ascribed solely to formation of gap junction channels and thought to be mediated by the transfer of small molecules between neighboring cells. In conflict with this hypothesis here we demonstrate that Cx43 can affect cell growth independently of gap junction formation. This conclusion is based on four findings: (1) There is a lack of correlation between the action of Cx43 mutants Cx43-S255A, Cx43-S279A, and Cx43-S282A on growth and cell coupling in 3T3 A31 fibroblasts. (2) Blockade of gap junction formation, by either heptan-1-ol treatment or culturing cells at low density, had no effect on the ability of the Cx43 mutants to control growth. (3) Wildtype Cx43 inhibited growth of Neuro2a cells under conditions where gap junctions were unable to form. (4) The CT domain of Cx43, which lacks intrinsic gap junction activity, is as effective as the wildtype molecule in suppressing the growth of Neuro2a cells. These observations demonstrate that Cx43 has dual functions: first, its well-accepted role in forming a gap junction channel and, second, a direct action of the connexin protein on growth that is mediated via the cytoplasmic carboxyl domain.     

Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle

Summary Gap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels     

C18:3-GM1a induces apoptosis in Neuro2a cells: enzymatic remodeling of fatty acyl chains of glycosphingolipids

GM1a [Gal beta1-3GalNAc beta1-4(NeuAc alpha2-3)Gal beta1-4Glc beta1-1Cer] is known to support and protect neuronal functions. However, we report that alpha-linolenic acid-containing GM1a (C18:3-GM1a), which was prepared using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase, induced apoptosis in neuronal cells. Intranucleosomal DNA fragmentation, chromatin condensation, and caspase activation, all typical features of apoptosis, were observed when mouse neuroblastoma Neuro2a cells were cultured with C18:3-GM1a but not GM1a containing stearic acid (C18:0) or oleic acid (C18:1). The phenotype of Neuro2a cells induced by C18:3-GM1a was similar to that evoked by lyso-GM1a. However, lyso-GM1a caused a complete disruption of lipid microdomains of Neuro2a cells and hemolysis of sheep erythrocytes, whereas C18:3-GM1a did neither. C18:3-GM1a, but not lyso-GM1a, was found to be abundant in lipid microdomains after the removal of loosely bound GM1a by BSA. The activation of stress-activated protein kinase/c-Jun N-terminal kinase in Neuro2a cells was observed with lyso-GM1a but not C18:3-GM1a. These results indicate that the mechanism of apoptosis induced by C18:3-GM1a is distinct from that caused by lyso-GM1a. This study also clearly shows that fatty acid composition of gangliosides significantly affected their pharmacological activities when added to the cell cultures and suggests why naturally occurring gangliosides do not possess polyunsaturated fatty acids as a major constituent.     

Suppression of human prostate cancer cell growth by forced expression of connexin genes

The cell-to-cell channels in gap junctions, formed of proteins called connexins (Cxs), provide a direct intercellular pathway for the passage of small signaling molecules (< or = 1 kD) between the cytoplasmic interiors of adjoining cells. It has been proposed that alteration in the expression and function of Cxs may be one of the genetic changes involved in the initiation of neoplasia. To elucidate the role of Cxs in the pathogenesis of human prostate cancer (PCA), the pattern of expression of Cx alpha 1 (Cx43) and Cx beta 1 (Cx32) was studied by immunocytochemical analysis in normal prostate and in prostate tumors of different histological grades. While normal prostate epithelial cells expressed only Cx beta 1, both Cx alpha 1 and Cx beta 1 were detected in PCA cells. The Cxs were localized at the cell-cell contact areas in normal prostate and well-differentiated prostate tumors; however, as prostate tumors progressed to more undifferentiated stages, the Cxs were localized in the cytoplasm, followed by an eventual loss in advanced stages. Thus, epithelial cells from prostate tumors showed subtle and gross alterations with regard to expression of Cx alpha 1 and Cx beta 1 and their assembly into gap junctions during the progression of PCA. Retroviral-mediated transfer of Cx alpha 1 and Cx beta 1 into a Cx-deficient human PCA cell line, LNCaP, inhibited growth, retarded tumorigenicity, and induced differentiation, and these effects were contingent upon the formation of gap junctions. In addition, the capacity to form gap junctions in most Cx-transduced LNCaP cells was lost upon serial passage. Taken together, these findings indicate that the control of proliferation and differentiation of epithelial cells in prostate tumors may depend on the appropriate assembly of Cx beta 1 and Cx alpha 1 into gap junctions and that the development of PCA may involve the positive selection of cells with an impaired ability to form gap junctions.     

Spatiotemporal expression of three gap junction gene products involved in fetomaternal communication during rat pregnancy.

The expression of three different members of the gap junction multigene family, alpha 1 (Cx43), beta 1 (Cx32), and beta 2 (Cx26), was analysed in the rat implantation chamber (a structural unit containing fetal, extraembryonic and maternal components within the pregnant uterus) during mid- and late stages of gestation as well as in the delivering, post-partum and non-pregnant uterus. A differential, spatiotemporal and cell-type-specific regulation of gap junctional coexpression was observed for beta 1 and beta 2 in all epithelia examined (visceral, luminal and glandular), as well as for alpha 1 and beta 2 in decidual cells and keratinocytes of the fetal epidermis. alpha 1 antigen was detected in the mesometrial stroma, mesometrial myometrium, connective tissue, mesothelia of the amnion and visceral yolk sac and in the allantoic mesodermal layer throughout gestation. In addition, expression of alpha 1 in the placental basal zone and trophoblast giant cells coincided with the differentiation of these cells. beta 2 expression was observed prominently in the chorionic villi of the placental labyrinth. The presence of beta 1 and beta 2 in the visceral epithelium (visceral yolk sac = the primary route for embryonic nourishment prior to the formation of the chorioallantoic placenta) and beta 2 in the chorionic villi (placental barrier = the major fetomaternal exchange route) suggests that gap junctions have an important role in fetomaternal communication.     

Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

Background

The gap junction protein, Connexin32 (Cx32), is expressed in various tissues including liver, exocrine pancreas, gastrointestinal epithelium, and the glia of the central and peripheral nervous system. Gap junction-mediated cell-cell communication and channel-independent processes of Cx32 contribute to the regulation of physiological and cellular activities such as glial differentiation, survival, and proliferation; maintenance of the hepatic epithelium; and axonal myelination. Mutations in Cx32 cause X-linked Charcot–Marie–Tooth disease (CMT1X), an inherited peripheral neuropathy. Several CMT1X causing mutations are found in the cytoplasmic domains of Cx32, a region implicated in the regulation of gap junction assembly, turnover and function. Here we investigate the roles of acetylation and ubiquitination in the C-terminus on Cx32 protein function. Cx32 protein turnover, ubiquitination, and response to deacetylase inhibitors were determined for wild-type and C-terminus lysine mutants using transiently transfected Neuro2A (N2a) cells.

Results

We report here that Cx32 is acetylated in transfected N2a cells and that inhibition of the histone deacetylase, HDAC6, results in an accumulation of Cx32. We identified five lysine acetylation targets in the C-terminus. Mutational analysis demonstrates that these lysines are involved in the regulation of Cx32 ubiquitination and turnover. While these lysines are not required for functional Cx32 mediated cell-cell communication, BrdU incorporation studies demonstrate that their relative acetylation state plays a channel-independent role in Cx32-mediated control of cell proliferation.

Conclusion

Taken together these results highlight the role of post translational modifications and lysines in the C-terminal tail of Cx32 in the fine-tuning of Cx32 protein stability and channel-independent functions.

     

Multiple gap junction genes are utilized during rat skin and hair development.

The expression of four different gap junction gene products (alpha 1, beta 1, beta 2, and beta 3) has been analysed during rat skin development and the hair growth cycle. Both alpha 1 (Cx43) and beta 2 (Cx26) connexins were coexpressed in the undifferentiated epidermis. A specific, developmentally regulated elimination of beta 2 expression was observed in the periderm at E16. Coinciding with the differentiation of the epidermis, differential expression of alpha 1 and beta 2 connexins was observed in the newly formed epidermal layers. alpha 1 connexin was expressed in the basal and spinous layers, while beta 2 was confined to the differentiated spinous and granular layers. Large gap junctions were present in the basal layer, while small gap junctions, associated with many desmosomes, were typical for the differentiated layers. Although the distribution pattern for alpha 1 and beta 2 expression remained the same in the neonatal and postnatal epidermis, the RNA and protein levels decreased markedly following birth. Hair follicle development was marked by expression of alpha 1 connexin in hair germs at E16. Following beta 2 detection at E20, the expression increased for both alpha 1 and beta 2 in developing follicles. A cell-type-specific expression was detected in the outer root sheath, in the matrix, in the matrix-derived cells (inner root sheath, cortex and medulla) and in the dermal papilla. In addition, alpha 1 was specifically expressed in the arrector pili muscle, while sebocytes expressed both alpha 1 and beta 3 (Cx31) connexin. beta 1 connexin (Cx32) was not detected at any stage analysed. The results indicate that multiple gap junction genes contribute to epidermal and follicular morphogenesis. Moreover, based on the utilization of gap junctions in all living cells of the surface epidermis, it appears that the epidermis may behave as a large communication compartment that may be coupled functionally to epidermal appendages (hair follicles and sebaceous glands) via gap junctional pathways.     

Immunotherapy of neuroblastoma by an Interleukin-21-secreting cell vaccine involves survivin as antigen

AIM: IL-21 is the most recently identified member of the IL-2 cytokine family. Here we studied the therapeutic efficacy of IL-21-gene-modified cells (Neuro2a/IL-21) in a syngeneic metastatic neuroblastoma (NB) model. MATERIALS AND METHODS: Neuro2a/IL-21 cells were tested as subcutaneous (sc) vaccine both in prophylactic and therapeutic settings. Depletion studies, cytotoxicity assay and immunohistochemical analyses were carried out to evaluate the mechanisms involved in tumor rejection. RESULTS: When injected sc in syngeneic A/J mice viable Neuro2a/IL-21 cells were rejected and induced resistance to a subsequent iv challenge with Neuro2a parental cells (Neuro2a/pc), suggesting the involvement of an immune response. More importantly, in mice bearing Neuro2a/pc micrometastases, a single sc injection of Neuro2a/IL-21 cells significantly increased the mean tumor-free survival of treated animals (43 vs. 22 days) and cured 14% of them. The administration of two or three doses of Neuro2a/IL-21 cell vaccine further increased the mean survival time to 54 and 75 days, and the cure rate to 27 and 33%, respectively, whereas the use of unmodified Neuro2a or mock-transfected cells had no effect. In vivo cell subset depletion and a Winn-assay indicated the involvement of CD8 + CTLs. Immunohistochemical analysis indicated a reduction of CD31+ and VEGFR2+ microvessels in late metastases from therapeutically vaccinated mice. A role of survivin as antigen was suggested by in vitro assays using survivin-synthetic CTL-epitopes. CONCLUSIONS: Our present data indicate that IL-21-secreting NB cells are effective as therapeutic vaccine in mice bearing metastatic NB, through a specific CTL response involving survivin as antigen, and suggest a potential interest for IL-21 in NB immuno-gene therapy.     

Virtual cloning,functional expression,and gating analysis of human connexin31.9

We have identified a novel gap junctiongene by searching the human genome sequence database that encodes aprotein designated as connexin31.9 (Cx31.9). Cx31.9 was most homologousto human Cx32.4 and did not cluster with either the purported - or-connexin subfamilies. Expression of Cx31.9 was detected by RT-PCRin human mRNA from several tissues including cerebral cortex, heart,liver, lung, kidney, spleen, and testis. A partial Cx31.9 sequence was also represented in the human Expressed Sequence Tag database. Cx31.9formed intercellular channels in both paired Xenopus oocytes and transfected neuroblastoma N2A cells that were distinguished by anapparent low unitary conductance (12-15 pS) and a remarkable insensitivity to transjunctional voltage. In contrast, Cx31.9 channelswere gated by cytoplasmic acidification or exposure to halothane likeother connexins. Cx31.9 was able to form heterotypic channels with thehighly voltage-sensitive Xenopus Cx38 (XenCx38), whichprovides an opportunity to study gating in heterotypic channels formedby hemichannels (connexons) composed of connexins with widely divergentproperties. Thus Cx31.9 is a novel human connexin that forms channelswith unique functional properties.

     

Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate

Disruption of gap junctional communication (GJC) by various compounds, including growth factors and tumor promoters, is believed to be modulated by the phosphorylation of a gap junctional protein, connexin43 (Cx43). We have previously demonstrated a platelet-derived growth factor (PDGF)-induced blockade of GJC and phosphorylation of Cx43 in T51B rat liver epithelial cells expressing wild-type PDGF receptor beta (PDGFr beta). Both of these actions of PDGF required participation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Similar requirements of MAPK were suggested in the modulation of GJC by other agents, including epidermal growth factor (EGF) and lysophosphatidic acid (LPA). Since many of these agents activate additional protein kinases, our present study examined whether activation of MAPK was sufficient for Cx43 phosphorylation and GJC blockade. By utilizing a variety of MAPK activators, we now show that activation of MAPK is not always associated with either Cx43 phosphorylation or disruption of GJC, which suggests a requirement for additional factors. Furthermore, pretreatment with hydrogen peroxide (H2O2), a potent MAPK activator but inefficient GJC/Cx43 modulator, abrogated PDGF- or TPA-induced disruption of GJC. While a 5 min H2O2 pretreatment abolished both PDGF- and TPA-induced Cx43 phosphorylation and GJC blockade, a simultaneous H2O2 treatment interfered only with GJC closure but not with the phosphorylation of Cx43 induced by PDGF and TPA. This finding indicates that, in addition to the Cx43 phosphorylation step, inhibition of GJC requires interaction with other components. H2O2-mediated abrogation of PDGF/TPA signaling can be neutralized by the antioxidant N-acetylcysteine (NAC) or by the tyrosine kinase inhibitor genistein. Taken together, our results suggest that disruption of GJC is not solely mediated by either activated MAPK or Cx43 phosphorylation but requires the participation of additional kinases and regulatory components. This complex mode of regulation is perhaps essential for the proposed functional role of GJC.     

Lembehyne A, a spongean polyacetylene, induces neuronal differentiation in neuroblastoma cell.

Lembehyne A (LB-A), a spongean polyacetylene, induced neuronal cell differentiation in a neuroblastoma cell line, Neuro 2A. The LB-A treatment of Neuro 2A cells predominantly resulted in a morphological change with bipolar neurites. The acetylcholinesterase activity of Neuro 2A was also increased by the treatment of LB-A. Furthermore, the cell cycle of Neuro 2A cells was found to be specifically blocked at the G1 phase by LB-A. The structure-activity relationship study using the LB-A analogues revealed the importance of the terminal 1-yn-3-ol and unsaturated long-chain alkyl moieties for the neuronal differentiation activity of LB-A.     

Involvement of membrane protein GDE2 in retinoic acid-induced neurite formation in Neuro2A cells

We show that a glycerophosphodiester phosphodiesterase homolog, GDE2, is widely expressed in brain tissues including primary neurons, and that the expression of GDE2 in neuroblastoma Neuro2A cells is significantly upregulated during neuronal differentiation by retinoic acid (RA) treatment. Stable expression of GDE2 resulted in neurite formation in the absence of RA, and GDE2 accumulated at the regions of perinuclear and growth cones in Neuro2A cells. Furthermore, a loss-of-function of GDE2 in Neuro2A cells by RNAi blocked RA-induced neurite formation. These results demonstrate that GDE2 expression during neuronal differentiation plays an important role for growing neurites.     

Lithium chloride regulates connexin43 in skeletal myoblasts in vitro: possible involvement in Wnt/beta-catenin signaling

Gap junction channels composed of connexin43 (Cx43) are essential for normal myogenic differentiation and skeletal muscle regeneration. Here, the aim was to study whether lithium chloride (LiCl) could regulate Cx43 expression and gap junction channel function by mimicking the Wnt/beta-catenin pathway in primary myoblasts. Cx43 mRNA expression in myoblasts was up-regulated in response to 5 mM LiCl. The enhanced Cx43 protein expression resulting from treatment with 5 and 10 mM LiCl for 24 h increased gap-junctional coupling in myoblasts. However, no obvious changes were observed with 20 mM LiCl. Furthermore, chronic treatment with 10 mM LiCl decreased Cx43 protein expression compared with untreated cells. The authors showed that LiCl mimicked the active canonical Wnt/beta-catenin signaling by glycogen synthase kinase-3beta (GSK-3beta) inactivation and accumulation of the effector protein beta-catenin into the nucleus. These results suggest that LiCl regulates Cx43 expression in skeletal myoblasts in vitro partly by a Wnt/beta-catenin-dependent pathway.